This proposal will investigate the mechanisms by which Fanconi anemia (FA) proteins regulate cellular response to oxidative stress in the context of hematopoiesis. The process of FA disease progression is characterized by bone marrow failure (BMF), clonal proliferation of hematopoietic stem and progenitor (HSC/P) cells, and progression to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). While clinical observations have established a correlation between abnormal accumulation of ROS and FA BMF/leukemic evolution, the molecular pathways in which FA proteins function to modulate physiologic oxidative stress have not been defined. We recently found that FA BM cells accumulated high levels of ROS generated by inflammation and that FA HSC/P cells were extremely sensitive to ROS-induced hematopoietic suppression. We have also shown that abnormal accumulation of ROS played a critical role in the evolution of leukemic clones in FA mouse model. Our most recent identification of the FANCD2- FOXO3a complex and preliminary characterization of impaired anti-oxidant defense in primary BM cells from FA patients open new research opportunities to extend this project to the renewal period. In Aim 1, we will test the hypothesis that physiologic oxidative stress contributes to BMF and progression to clonal hematopoiesis in FA through influencing HSC/P cell proliferation and apoptosis, by assessing the effect of inflammatory ROS on proliferation and apoptosis of HSC/P cells from FA children at three different stages (BMF, MDS, and AML) of disease progression and in a humanized NOG/SGM3 mouse xenograft model. In Aim 2, we will test the hypothesis that functional interaction between the FA proteins and other cell signaling pathways play important roles in maintaining normal hematopoiesis under physiologic oxidative stress, with focus on major oxidative stress response pathways involving FOXO3a and cellular anti-oxidant defense systems. In Aim 3, we will test the hypothesis that increased inflammatory ROS and vulnerability of chromosomal DNA to oxidative damage would provide a potential genetic mechanism for FA genomic instability, with focus on the roles of FA proteins in oxidative DNA-damage response and repair, and the functional relationship between inflammatory ROS and genomic instability during FA leukemogenesis using two FA preleukemic models. The knowledge gained from these studies may lead to a new avenue of research designed to further explore the pathogenic role of oxidative stress not only in FA but also in cancer- related hematological diseases in general. In addition, new insights on the potential integration of the FA proteins in other oxidative-stress signaling pathways can suggest new targets for therapeutic prevention and treatment of BMF and cancer progression of these diseases.